Among the arthropod-borne flaviviruses, the four dengue virus serotypes (DEN1-4) that constitute a serologically distinct subgroup are most important in terms of human morbidity and geographic distribution. A safe and effective dengue vaccine is still not available. Passive immunization using monoclonal antibodies from humans or non-human primates represents an attractive alternative to prevention of dengue. Chimpanzee Fab antibody fragments were recovered by repertoire cloning of bone marrow mRNAs and analyzed for antigen binding specificity, VH heavy chain and VL light chain sequences and neutralizing activity against dengue virus in vitro. Several distinct Fab antibody fragments were identified from libraries constructed from chimpanzees that had been infected with DEN1, DEN2, DEN3 and DEN4. Radio-immunoprecipitation and binding competition by ELISA demonstrated that Fab antibodies reactive to DEN4 structural protein prM or E were recovered. Importantly, two related E-specific Fabs neutralized DEN4 very efficiently by an in vitro assay. One of the neutralizing Fabs was converted to a whole antibody by combination with human antibody sequences. The humanized chimpanzee antibody produced in mammalian CHO cells neutralized DEN4 strains from different geographic origins at a similar high neutralizing titer. Both the Fab fragment and the whole antibody showed a strong binding affinity for DEN4. This success prompted us to similarly identify chimpanzee Fab antibodies against each of the other three dengue virus serotypes. Several Fab monoclonal antibodies recovered in this manner are novel in that they cross-neutralized DEN1 and DEN2 efficiently. The humanized monoclonal antibody made from one of the selected Fab fragments proved to be also cross-neutralizing. These humanized monoclonal antibodies may prove valuable for passive immunization against dengue. Repertoire cloning and identification of neutralizing antibodies against other flaviviruses represent a logical expansion of the research.